Mg(II) binding by bovine prothrombin fragment 1 via equilibrium dialysis and the relative roles of Mg(II) and Ca(II) in blood coagulation

The first direct equilibrium dialysis titration of the blood coagulation protein bovine prothrombin fragment 1 with Mg(II) is presented. Fragment 1 has fewer thermodynamic binding sites for Mg(II) than Ca(II), less overall binding affinity, and significantly less cooperativity. Several nonlinear curve fitting models were tested for describing the binding of fragment 1 with Mg(II), Ca(II), and mixed metal binding data. The Mg(II) data is represented by essentially five equivalent, noninteracting sites; for Ca(II), a model with three tight, cooperative sites and four "loose", equal affinity, noninteracting sites provides the best model. Based on the reported equilibrium dialysis data and in conjunction with other experimental data, a model for the binding of Ca(II) and Mg(II) to bovine prothrombin fragment 1 is proposed. The key difference between the binding of these divalent ions is that Ca(II) apparently causes a specific conformational change reflected by the cooperativity observed in the Ca(II) titration. The binding of Ca(II) ions to the three tight, cooperative sites establishes a conformation that is essential for phospholipid X Ca(II) X protein binding. The filling of the loose sites with Ca(II) ions leads to charge reduction and subsequent phospholipid X Ca(II) X protein complex interaction. Binding of Mg(II) to bovine prothrombin fragment 1 does not yield a complex with the necessary phospholipid-binding conformation. However, Mg(II) is apparently capable of stabilizing the Ca(II) conformation as is observed in the mixed metal ion binding data and the synergism in thrombin formation.     

Stereochemical control over Mn(II)-thio versus Mn(II)-oxy coordination in adenosine 5'-O-(1-thiodiphosphate) complexes at the active site of creatine kinase

The stereochemical configurations of the Mn(II) complexes with the resolved epimers of adenosine 5'-O-(1-thiodiphosphate) (ADP alpha S), bound at the active site of creatine kinase, have been determined in order to assess the relative strengths of enzymic stereoselectivity versus Lewis acid/base preferences in metal-ligand binding. Electron paramagnetic resonance (EPR) data have been obtained for Mn(II) in anion-stabilized, dead-end (transition-state analogue) complexes, in ternary enzyme-MnIIADP alpha S complexes, and in the central complexes of the equilibrium mixture. The modes of coordination of Mn(II) at P alpha in the nitrate-stabilized, dead-end complexes with each epimer of ADP alpha S were ascertained by EPR measurements with (Rp)-[alpha-17O]ADP alpha S and (Sp)-[alpha-17O]ADP alpha S. The EPR spectrum for the complex with (Rp)-[alpha-17O]ADP alpha S showed inhomogeneous broadening due to unresolved superhyperfine coupling from coordinated 17O at P alpha. By contrast, the EPR spectrum for Mn(II) in complex with (Sp)-[alpha-17O]ADP alpha S is indistinguishable from that obtained for a matched sample with unlabeled (Sp)-ADP alpha S. A reduction in the magnitude of the 55Mn hyperfine coupling constant in the spectrum for the complex containing (Sp)-ADP alpha S is indicative of Mn(II)-thio coordination at P alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA-copper (II) complex and the DNA conformation

Spectrophotometric, sedimentation, infrared, optical rotatory dispersion (ORD), and circular dichroism (CD) methods have been used to demonstrate the structural changes in DNA induced by the interaction of copper(II) with bases and to elucidate the complex binding sites. As shown by the electrolyte-induced reversion (addition of salts) of temperature-denatured copper DNA the effectiveness of re-formation of the double-stranded structure depends on the temperature, copper(II) ion concentration, and on the base composition of the DNA. Exposure of heat-denatured copper DNA to higher temperatures decreases the reversion effect on addition of electrolyte. The results indicate that a greater fraction with a cooperative transition appears on heating DNA to 80 or 100°C at a Cu²⁺/DNA-P ratio of 2 : 1 than at a Cu²⁺/DNA-P ratio of 1 : 1. With AT-rich copper DNA, reversion to the native DNA structure was not observed. Selective methylation of guanine residues in DNA also affects the electrolyte-induced reversion, indicating the importance of GC pairs for copper(II) binding and the reversion to the native structure. Temperature-denatured copper DNA shows an increased sedimentation coefficient Which decreases again after electrolyte-induced reversion. This change in s is reduced by selective methylation of DNA. Complex formation between copper(II) and the bases is accompanied by a conformational change of the DNA double-helical structure as demonstrated by ORD and CD experiments. The ORD profile of GC-rich DNA is much more affected by copper(II) than that of AT-rich ones. Even at very low copper(II) concentrations, e.g., at 0.02 and 0.2 Cu²⁺/DNA-P, the ORD and CD measurements exhibit conformational changes of the DNA secondary structure at room temperature. By comparing the infrared spectra of deoxynucleosides with that of DNA of different GC content it has been shown that both guanine and cytosine are involved in the formation of the complex of copper(II) with DNA. N-7 and O at C-6 in guanine and N-3 as well as O of C-2 in cytosine are discussed as the most probable binding sites in DNA. A binding model for the coordination of the copper(II) ion between guanine and cytosine of the opposite strands is suggested. The results are in good agreement with the assumptions and predictions made by Eichhorn and Clark about the complexing of copper(II) with DNA. The recent proposal made by Schreiber and Daune about an interaction of the type guanine–Cu²⁺–guanine cannot be excluded as an additional kind of coordination of copper(II) in DNA.     

The role of the metal ion in the refolding of denatured bovine Co(II)-carbonic anhydrase II

Conditions for reactivation of guanidine-HCl-denatured bovine Co(II)-carbonic anhydrase II are given. The renaturation is accompanied by recovery of the native Co(II)-spectrum of the enzyme. After studying the kinetics of the renaturation process, the metal ion involvement in the refolding pathway can be summarized as follows: (1) Formation of an inactive Co(II)-intermediate with the metal ion firmly bound. No native Co(II)-spectrum is observed in this state, probably due to octahedral coordination of the metal ion in this intermediate. (2) Formation of an inactive Co(II)-intermediate with a native Co(II)-spectrum. The final tetrahedral coordination of the metal ion seems to have been formed in this state. (3) Formation of the active conformation of the enzyme. A functioning active-site is formed after some rearrangements of the polypeptide chain. This isomerisation step does not need to be preceded by formation of the intermediate with a native Co(II)-spectrum. Coordination of Co2+ in a native-like manner is, however, a prerequisite for enzymic activity. It is tentatively suggested that the metal ion is involved in stabilizing a nucleation structure formed at the bottom of the active centre. This probably occurs through binding of Co2+ to some or all of its histidyl ligands in this region after an early structuration of the metal ion binding site. The mechanisms of Co2+ appear to be similar for the refolding enzyme and the native apoenzyme, inferring that the binding site formed as a result of the nucleation process probably has the same structure as in the native conformation.     

Dihydroorotase from Escherichia coli. Substitution of Co(II) for the active site Zn(II).

Treatment of Escherichia coli dihydroorotase (a homodimer of subunit molecular weight 38,729) containing only the 1 active site Zn(II) ion per subunit with the sulfhydryl reagent N-(ethyl)-maleimide (NEM) blocks the two external Zn(II) sites per subunit and dramatically lessens the precipitation caused by high concentrations of Zn(II); stabilizes the enzyme partially against air oxidation and dilution inactivation; makes the active site Zn(II) easier to remove; and lowers Km and increases kcat. Treatment of NEM-blocked dihydroorotase ((NEM)dihydroorotase) with the chelator 2,6-pyridinedicarboxylic acid at pH 5.0 in the absence of oxygen and trace metal ions removes the active site Zn(II) with a half-life of 15 min, allowing the production of milligram amounts of moderately stable apo-(NEM)dihydroorotase in about 80% yield. Treatment of apo-(NEM)dihydroorotase with Co(II) at pH 7.0 produces (NEM)dihydroorotase completely substituted at the active site with Co(II) in 100% yield: analysis gives 0.95-1.1 g atoms of Co(II) per active site and 0.03-0.05 g atoms of Zn(II) per active site. This Co(II)-(NEM)dihydroorotase is hyperactive at pH 8. The electronic absorption spectrum of Co(II)-(NEM)dihydroorotase at pH 6.5 implicates an active site thiol group as a ligand to the metal ion. The spectrum is inconsistent with tetrahedral coordination of the active site metal ion and is most consistent with a pentacoordinate structure.     

The complexes of adenosine and organocobalamins with palladium (II)

The complexes of adenosine, methylcobalamin, adenosylcobalamin and the derivatives of adenosylcobalamin with tetrachloropalladate (II) are described. The compositions of the complexes were established by means of spectrophotometric titration. Depending on the structure of organocobalamins, three types of complexes with tetrachloropalladate (II) are formed. In the case of methylcobalamin, the palladium (II) is coordinated at the “lower” benzimidazole ligand. In adenosylcobalamin, palladium (II) coordinates the “upper” adenosyl ligand. The N-7-position in adenosine ligand and N-3-position in 5,6-dimethylbenzimidazolylribotide are the most probable coordination sites of palladium (II).     

Europium(III) luminescence and tyrosine to terbium(III) energy-transfer studies of invertebrate (octopus) calmodulin.

The effects of minor differences in the amino acid sequences between a vertebrate (bovine testes) and an invertebrate (octopus) calmodulin on metal ion binding were investigated via laser-induced Eu3+ and Tb3+ luminescence. Amino acid substitutions at residues which are coordinated to the metal ion do not produce any detectable changes in the 7F0----5D0 excitation spectrum of the Eu3+ ion bound to octopus calmodulin relative to bovine testes calmodulin; only minor differences in the excited-state lifetime values in D2O solution are observed. The dissociation constants for Eu3+ (1.0 +/- 0.2 microM) and Tb3+ (5 +/- 1 microM) from the weak lanthanide binding sites (III and IV, numbered from the amino terminus) of octopus calmodulin were measured using luminescence techniques. Both values agree well with those reported previously for bovine testes calmodulin [Mulqueen, P. M., Tingey, J. M., & Horrocks, W. D., Jr. (1985) Biochemistry 24, 6639-6645]. The measured dissociation constant of Eu3+ bound in the tight lanthanide binding sites (I and II) is 6 +/- 2 nM for octopus calmodulin and 12 +/- 2 nM for bovine testes calmodulin. The distances between sites I and II (12.4 +/- 0.5 A) and sites III and IV (11.7 +/- 0.8 A) were determined from F?rster-type energy transfer in D2O solutions of octopus calmodulin containing bound Eu3+ donor and Nd3+ acceptor ions. F?rster theory parameters for nonradiative energy transfer between Tyr138 and Tb3+ ions bound at sites III and IV of octopus calmodulin were comprehensively evaluated, including a dynamics simulation of the orientation factor kappa 2. This theory is found to account quantitatively for the observed energy-transfer efficiency as evaluated from the observed sensitized Tb3+ emission.     

Lanthanide spectroscopic studies of the dinuclear and Mg(II)-dependent PvuII restriction endonuclease

Type II restriction enzymes are homodimeric systems that bind four to eight base pair palindromic recognition sequences of DNA and catalyze metal ion-dependent phosphodiester cleavage. While Mg(II) is required for cleavage in these enzymes, in some systems Ca(II) promotes avid substrate binding and sequence discrimination. These properties make them useful model systems for understanding the roles of alkaline earth metal ions in nucleic acid processing. We have previously shown that two Ca(II) ions stimulate DNA binding by PvuII endonuclease and that the trivalent lanthanide ions Tb(III) and Eu(III) support subnanomolar DNA binding in this system. Here we capitalize on this behavior, employing a unique combination of luminescence spectroscopy and DNA binding assays to characterize Ln(III) binding behavior by this enzyme. Upon excitation of tyrosine residues, the emissions of both Tb(III) and Eu(III) are enhanced severalfold. This enhancement is reduced by the addition of a large excess of Ca(II), indicating that these ions bind in the active site. Poor enhancements and affinities in the presence of the active site variant E68A indicate that Glu68 is an important Ln(III) ligand, similar to that observed with Ca(II), Mg(II), and Mn(II). At low micromolar Eu(III) concentrations in the presence of enzyme (10-20 microM), Eu(III) excitation (7)F(0) --> (5)D(0) spectra yield one dominant peak at 579.2 nm. A second, smaller peak at 579.4 nm is apparent at high Eu(III) concentrations (150 microM). Titration data for both Tb(III) and Eu(III) fit well to a two-site model featuring a strong site (K(d) = 1-3 microM) and a much weaker site (K(d) approximately 100-200 microM). Experiments with the E68A variant indicate that the Glu68 side chain is not required for the binding of this second Ln(III) equivalent; however, the dramatic increase in DNA binding affinity around 100 microM Ln(III) for the wild-type enzyme and metal-enhanced substrate affinity for E68A are consistent with functional relevance for this weaker site. This discrimination of sites should make it possible to use lanthanide substitution and lanthanide spectroscopy to probe individual metal ion binding sites, thus adding an important tool to the study of restriction enzyme structure and function.     

Coordination mode of adenosine 5'-diphosphate in ternary systems containing Cu(II), Cd(II) or Hg(II) ions and polyamines

The interactions of adenosine 5'-diphosphate (ADP) with some polyamines (PA) (1,3-diaminopropane (tn), 1,4-diaminobutane (Put), 1,7-diamino-4-azaheptane (3,3-tri) and 1,8-diamino-4-azaoctane (Spd)) both in presence and in the absence of metal ions (Cu(II), Cd(II) and Hg(II)) have been studied. In the metal-free systems the formation of adducts (ADP)Hx(PA) has been observed, in which the main reaction centres are the endocyclic nitrogen atoms of the purine ring, the phosphate group of the nucleotide and the protonated nitrogen atoms of the polyamine. The effectiveness of the phosphate group in formation of adducts has been found to decrease in the series Put > Spd > Spm and to be lower than in the reactions with shorter homologues of biogenic amines. In the ternary systems with metal ions the formation of molecular complexes (ML L' type) has been evidenced in which the protonated polyamine interacts with the nitrogen atoms N(1) or N(7) of the purine ring of the nucleotide. In the ternary systems Cu(II)/ADP/polyamine the coordination dichotomy observed in the binary system Cu(II)/ADP disappears. In the systems with Hg(II) ions the pH range of the dichotomy is extended, while for the systems Cd(II)/ADP/polyamine no changes of the range relative to the binary system Cd(II)/ADP have been noted.     

Electron paramagnetic resonance studies of MN(II) complexes with myosin subfragment 1 and oxygen 17-labeled ligands

Ligands in the first coordination sphere of Mn(II) in the complex of MnADP with myosin subfragment 1 from rabbit skeletal muscle have been investigated. EPR spectroscopy was used to detect superhyperfine coupling between unpaired electrons of the metal ion and the nuclei of oxygen atoms specifically labeled with oxygen 17. The results show that ADP is a beta-monodentate ligand for Mn(II) and that there are probably two water oxygens directly bound to Mn(II). The inhibitory complex of vanadate with subfragment 1 . MnADP was also investigated. Vanadate-dependent changes in the EPR spectra for enzyme-bound Mn(II) indicate that the coordination sphere of MN(II) changes upon binding of vanadate. ADP remains a beta-monodenate ligand in the complex and experiments with 17O-labeled water indicate that two oxygen atoms originally in water are ligands in the complex. However, the oxygens of vanadate equilibrate with those of water during sample preparation so that one of these ligands may be a vanadate oxygen. Three additional ligands, probably from the protein, are required to complete the sextet of ligands to Mn(II) in both complexes studied.     

Glutamine synthetase from Salmonella typhimurium: manganese(II), substrate, and inhibitor interaction with the unadenylylated enzyme.

Unadenylylated glutamine synthetase (EC 6.3.1.2) was isolated and purified to homogeneity from Salmonella typhimurium. The enzyme molecule is a symmetrical aggregate of 12 subunits arranged in two hexagonal layers, as is evident from electron micrographs. The subunit molecular weight of the enzyme was found to be approximately 50,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate when compared to Escherichia coli glutamine synthetase and other protein standards. A long tube of glutamine synthetase was formed as a single-stranded coil resulting from incubation of the enzyme in a low ionic strength buffer. A study of Mn(II) binding to the unadenylylated enzyme at 25 °C was conducted as a function of pH. At pH 7.1 two classes of metal ion sites per subunit were found with K_D values of 3.7 × 10^?6 and 1.7 × 10^?4m, while at pH 6.8 these values were 1.1 × 10^?5 and 1.0 × 10^?4m, respectively. Only one set of binding sites was observed at pH 6.2 with a K_D value of 1.0 × 10^?4m. The metal ion binding sites were further investigated by monitoring proton relaxation rates (prr) and the epr spectrum of enzyme-bound Mn(II). The longitudinal prr of water protons at pH 7.1 indicate that protons interacting with enzyme-Mn(II) at the “tight” site (K_D = 3.7 × 10^?6) are de-enhanced (?_b1 = 0.42) and result from water protons beyond the inner coordination sphere. The second Mn(II) site has a value of ?_b2 = 35 for the binary enhancement, suggesting that this site probably has two to three rapidly exchanging water molecules in its coordination sphere. The epr spectrum of enzyme-bound Mn(II) at the “tight” site is isotropic and is dramatically sharpened by adding the substrate analog methionine sulfoximine. Subsequent addition of ATP or the ATP analog, AMP-PCP (adenylyl methylene diphosphate) produced anisotropic spectra that were similar, suggesting that both ATP and AMP-PCP bind similarly on the enzyme surface. However, a marked change in the Mn(II) environment from anisotropic to near cubic results from the addition of ADP to the quaternary enzyme-Mn(II)-sulfoximine- (AMP-PCP) complex, indicating that ADP displaces AMP-PCP. No change in the anisotropic spectrum due to the enzyme-Mn(II)-sulfoximine-ATP complex is seen by the addition of ADP. This experimental result supports the experimental findings of Ronzio and Meister [Proc. Nat. Acad. Sci. USA59, 164 (1968)], who established that ATP phosphorylates methionine sulfoximine, thereby producing an inactive enzyme. The allosteric effectors, AMP and Trp, have little effect on the epr spectrum of the complex formed from Mn(II), enzyme, sulfoximine, and ADP, suggesting the absence of direct coordination of AMP or Trp to the bound Mn(II). The prr and epr results reported herein with glutamine synthetase from S. typhimurium when compared to those seen for the enzyme from E. coli [Villafranca et al., Biochemistry15, 544 (1976)] demonstrate some similarities but also many substantial differences between the enzymes from these two bacterial sources.     

Coordination scheme and stereochemical configuration of manganese(II) adenosine 5'-diphosphate at the active site of 3-phosphoglycerate kinase

The structure of the MnIIADP complex at the active site of 3-phosphoglycerate kinase from yeast has been investigated by electron paramagnetic resonance (EPR) spectroscopy. Inhomogeneous broadening in the EPR signals for Mn(II) resulting from unresolved superhyperfine coupling to 17O regiospecifically incorporated into ADP shows that Mn(II) is coordinated to the alpha- and beta-phosphate groups of ADP at the active site of the enzyme. The EPR pattern for the enzyme-MnIIADP complex is characteristic of a predominantly axially symmetric zero-field splitting tensor. The symmetry and magnitude of the zero-field splitting interaction suggest that there is an additional negatively charged oxygen ligand in the coordination sphere of Mn(II). EPR measurements for solutions of the enzyme-MnIIADP complex in 17O-enriched water indicate that there are also two or three water molecules in the coordination sphere of the metal ion. EPR data for complexes with the two epimers of [alpha-17O]ADP have been used to determine the stereochemical configuration of the MnIIADP complex at the active site. EPR spectra for Mn(II) in the enzymic complex with (Rp)-[alpha-17O]ADP show an inhomogeneous broadening due to superhyperfine coupling with 17O whereas spectra for (Sp)-[alpha-17O]ADP complexes are indistinguishable from those for matched samples with unlabeled ADP. These results show that 3-phosphoglycerate kinase selectivity binds the alpha configuration of the alpha, beta chelate of MnIIADP. Addition of 3-phosphoglycerate to form the dead-end complex (enzyme-MnIIADP-3-phosphoglycerate) does not alter the EPR spectrum, but addition of vanadate to this complex causes marked changes in the spectral parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural mechanism of ribonucleotide discrimination by a Y-family DNA polymerase

The ability of DNA polymerases to differentiate between ribonucleotides and deoxribonucleotides is fundamental to the accurate replication and maintenance of an organism's genome. The active sites of Y-family DNA polymerases are highly solvent accessible, yet these enzymes still maintain a high selectivity towards deoxyribonucleotides. Here, we biochemically demonstrate that a single active-site mutation (Y12A) in Dpo4, a model Y-family DNA polymerase, causes both a dramatic loss of ribonucleotide discrimination and a decrease in nucleotide incorporation efficiency. We also determined two ternary crystal structures of the Dpo4 Y12A mutant incorporating either dATP or ATP nucleotides opposite a template dT base. Interestingly, both dATP and ATP were hydrolyzed to dADP and ADP, respectively. In addition, the dADP and ADP molecules adopt a similar conformation and position at the polymerase active site to a ddADP molecule in the ternary crystal structure of wild-type Dpo4. The Y12A mutant loses stacking interactions with the deoxyribose of dNTP, which destabilizes the binding of incoming nucleotides. The mutation also opens a space to accommodate the 2′-OH group of the ribose of NTP in the polymerase active site. The structural change leads to the reduction in deoxynucleotide incorporation efficiency and allows ribonucleotide incorporation.     

Volume changes in the binding of lanthanides to peptide analogues of loop II of calmodulin

The solution expansion accompanying coordination of lanthanide ions to synthetic peptide analogues of a metal-binding loop in calmodulin was determined by a density method. This study was designed to further test the hypothesis that the nonlinear expansions observed upon sequential addition of Ca2+ to intracellular calcium-binding proteins reflect principally upon the coordination event at specific binding sequences. Three peptides of 13 residues each were synthesized as analogues of binding loop II in mammalian calmodulin: Peptide I was the native analogue; peptide II contained an aspartyl in place of an asparaginyl residue at position 5 from the N-terminus; for peptide III, the aspartyl residue in position 3 of the native analogue was interchanged with the asparaginyl residue in position 5. Thus, the number of charged-oxygen donor atoms for coordination was the same in I and in III, but the latter peptide could permit two pairs of acidic groups to converge toward the metal ion as in some loops of these proteins. The observed expansions with different lanthanide ions to the same peptide varied appreciably, suggesting dissimilar structures [Gariépy et al. (1983) Biochemistry 22, 1765-1772]; coordination to the simpler tetracarboxylate sequestrants, on the other hand, generated an expansion profile approximately as expected from the properties of the lanthanide series. The largest expansions were generated with peptide II (having the additional acidic group) for all lanthanides tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acid-base properties and copper(II) complexes of dipeptides containing histidine and additional chelating bis(imidazol-2-yl) residues

Copper(II) complexes of dipeptides of histidine containing additional chelating bis(imidazol-2-yl) agent at the C-termini (PheHis-BIMA [N-phenylalanyl-histidyl-bis(imidazol-2-yl)methylamine] and HisPhe-BIMA [N-histidyl-phenylalanyl-bis(imidazol-2-yl)methylamine]) were studied by potentiometric, UV-Visible and Electron Paramagnetic Resonance (EPR) techniques. The imidazole nitrogen donor atoms of the bis(imidazol-2-yl)methyl group are described as the primary metal binding sites forming stable mono- and bis(ligand) complexes at acidic pH. The formation of a ligand-bridged dinuclear complex [Cu2L2]4+ is detected in equimolar solutions of copper(II) and HisPhe-BIMA. The coordination isomers of the dinuclear complex are described via the metal binding of the bis(imidazol-2-yl)methyl, amino-carbonyl and amino-imidazole(His) functions. In the case of the copper(II)-PheHis-BIMA system the [NH2, N-(amide), N(Im)] tridentate coordination of the ligand is favoured and results in the formation of di- and trinuclear complexes [Cu2H(-1)L]3+ and [Cu3H(-2)L2]4+ in equimolar solutions. The presence of these coordination modes shifts the formation of "tripeptide-like" ([NH2, N-, N-, N(Im)]-coordinated) [CuH(-2)L] complexes into alkaline pH range as compared to other dipeptide derivatives of bis(imidazol-2-yl) ligands. Although there are different types of imidazoles in these ligands, the deprotonation and coordination of the pyrrole-type N(1)H groups does not occur below pH 10.     

X-ray crystallographic analyses of complexes between bovine beta-trypsin and Schiff base copper(II) or iron(III) chelates

To establish the structural basis underlying the activity of a novel series of metal-chelate trypsin inhibitors, the structures of p-amidinosalicylidene-l-alaninato(aqua)copper(II) (1a), m-amidinosalicylidene-l-alaninato(aqua)copper(II) (1b), bis(p-amidinosalicylidene-l-alaninato)iron(III) (2a), and bis(m-amidinosalicylidene-l-alaninato)iron(III) (2b) bound to bovine beta-trypsin were studied by X-ray crystallography. The amidinium group of the inhibitor donates hydrogen bonds to Asp189, Gly219 and Ser190, as seen before in trypsin-benzamidine complexes. The copper(II) ion of 1a is situated away from trypsin's catalytic triad residues, and is octahedrally coordinated by a Schiff base and three water molecules. In contrast, the copper(II) ion of 1b is situated close to the catalytic triad and adopts a square pyramidal coordination geometry. The iron(III) ion of 2a is octahedrally coordinated by two Schiff base ligands and, like the copper(II) ion of 1a, is situated away from the catalytic triad. The p-amidinophenyl ring of a second Schiff base ligand of 2a is directed toward a hydrophobic groove formed by Trp215 and Leu99. Finally, the iron(III) ion of 2b appears to be replaced by magnesium(II), which is octahedrally coordinated by a Schiff base, Gln192 and two water molecules. One of the Schiff base ligands seen in the trypsin-2a complex or in the unbound form of 2b is replaced by water molecules and Gln192. His57 and Ser195 form water-mediated interactions with the magnesium(II) ion of 2b, and Ser195 also forms a hydrogen bond with the phenolic oxygen atom of the Schiff base ligand. These structures reveal a novel mode of interaction between metal-chelate inhibitors and serine proteases, thus providing a structural basis for the development of more potent inhibitors against a variety of trypsin-like enzymes.     

Macromolecularization of a tripeptide analogue of the Cu(II) binding site of human serum albumin: 2. Conformational and binding properties towards Cu(II) and Ni(II)ions of Gly-Gly-His derivatives of poly(l-lysine)

The conformational and binding properties towards Cu(II) and Ni(II) ions of Gly-Gly-His derivatives of poly(l-lysine) have been investigated mainly using circular dichroism (c.d.) spectroscopy. These derivatized polymers can be considered macromolecular analogues of the Cu(II) and Ni(II) binding site of human serum albumin. It has been shown that modification up to 53% of the ε-amino groups of lysine side chains by covalent binding of the tripeptide unit Gly-Gly-His does not induce appreciable alteration of the α-helix forming tendency of the polylysine backbone. The derivatized polymers exhibit strong affinity towards Cu(II) and Ni(II) ions. At neutral pH, complexes are formed in which each tripeptide chelating unit is linked to one metal ion. The spectral characteristics in the visible absorption region are consistent with a square planar geometry of the complexes, with deprotonated peptide groups and one imidazole nitrogen in the coordination sphere of the ion. C.d. measurements in the far u.v. indicate that complex formation in the side chains causes an increase of ordered structure of the peptide backbone at neutral pH. This fact is interpreted in terms of a reduced electrostatic repulsion among side chains due to charge neutralization in the tripeptide units linked to metal ions.     

Strong protective action of Copper(II) N-substituted sulfonamide complexes against reactive oxygen species

Copper(II) complexes of N-benzothiazolsulfonamides, [Cu(N-2-(5,6-dimethylbenzothiazole)toluenesulfonamidate)(2)(dmso)(2)] (1), [Cu(N-2-(6-chlorobenzothiazole)benzenesulfonamidate)(2)(dmso)(2)] (2) and [Cu(N-2-(6-chlorobenzothiazole)toluenesulfonamidate)(2)(dmso)(2)] (3) with interesting protective properties against superoxide radicals have been prepared. The compounds have been characterized by X-ray diffraction and their chemical properties have been studied by spectroscopic methods. The crystal structure of 1 shows that the copper(II) is surrounded by two benzothiazole N atoms from the sulfonamide ligands and two O atoms from the dimethylsulfoxide molecules in a square planar arrangement. The coordination polyhedron around copper(II) in 2 and 3 is distorted square pyramidal being the metal ion linked to benzothiazole N and sulfonamidate O atoms of the ligand and to two dimethylsulfoxide O atoms. The three complexes have a strong protective action over Delta sod1 mutant of Saccharomyces cerevisiae against reactive oxygen radicals derived from respiration and against those generated by hydrogen peroxide and menadione.     

Macromolecularization of a tripeptide analogue of the Cu(II) binding site of human serum albumin: 2. Conformational and binding properties towards Cu(II) and Ni(II)ions of Gly-Gly-His derivatives of poly(l-lysine)

The conformational and binding properties towards Cu(II) and Ni(II) ions of Gly-Gly-His derivatives of poly(l-lysine) have been investigated mainly using circular dichroism (c.d.) spectroscopy. These derivatized polymers can be considered macromolecular analogues of the Cu(II) and Ni(II) binding site of human serum albumin. It has been shown that modification up to 53% of the ε-amino groups of lysine side chains by covalent binding of the tripeptide unit Gly-Gly-His does not induce appreciable alteration of the α-helix forming tendency of the polylysine backbone. The derivatized polymers exhibit strong affinity towards Cu(II) and Ni(II) ions. At neutral pH, complexes are formed in which each tripeptide chelating unit is linked to one metal ion. The spectral characteristics in the visible absorption region are consistent with a square planar geometry of the complexes, with deprotonated peptide groups and one imidazole nitrogen in the coordination sphere of the ion. C.d. measurements in the far u.v. indicate that complex formation in the side chains causes an increase of ordered structure of the peptide backbone at neutral pH. This fact is interpreted in terms of a reduced electrostatic repulsion among side chains due to charge neutralization in the tripeptide units linked to metal ions.     

A circular dichroic study of Cu(II) binding to bovine alpha-lactalbumin.

The visible and ultraviolet circular dichroic spectra resulting from the interaction of bovine alpha-lactalbumin with successive Cu(II) ions have been recorded under a variety of conditions. Analysis of the observed change-transfer and d-d band transitions can be made in terms of two kinds of binding sites: at a histidyl group and at the N-terminal amino group, respectively. At basic pH the amide nitrogens of the peptide backbone progressively take part in the coordination. The occupation of the high affinity calcium binding site by Ca(II) and Mn(II) does not influence the Cu(II) binding process, suggesting that there is no direct interaction between this site and the Cu(II) binding sites.